Biomaterials and bioengineering are fast growing areas that have become critical parts of modern medicine. Because of their versatility and propensity to form arrays, viral protein cages are ideal substrates to build presentation systems. Through genetic engineering, the self-assembled viral structural protein is an outstanding platform for biomaterial synthesis and a scaffold for integration of foreign molecules in designed patterns, which can be used as vaccines or drugs.
A recent example using viral particles for antigen presentation is the chimera of flock house virus (FHV) VLP with a 180-amino acid antigen insertion of Bacillus anthracis, described by Manayani et al. The recombinant virus-like particles function both as an anthrax antitoxin and as a molecular scaffold for an anthrax vaccine, and combine the functions of an anthrax antitoxin and vaccine in a single compound. Another example is the Cowpea mosaic virus (CPMV), described by Chatterji et al., which includes the use of Cowpea mosaic virus for both epitope presentation and as a matrix for the attachment of peptides and proteins. Entire proteins have been chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral CPMV particles. Also, a hepatitis B virus (HBV) capsid-like particle (CLP) containing a surface antigen (OspA) of Borrelia burgdorferi has been described.
Norovirus (NOR), also known previously as “Norwalk-Like Virus” (NLV) or small round structured virus, is the most important viral pathogen of epidemic acute gastroenteritis that occurs in both developed and developing countries. These genetically diverse viruses comprise two major genogroups (GI and GII) and approximately 30 genotypes. NORs belong to the Caliciviridae family and are icosahedral, single stranded, positive-sense RNA viruses whose capsids are composed of 180 copies of a single major structural protein.
In the past, the biological characterization of human NORs had been hampered because the virus failed to grow in cell cultures efficiently and no suitable animal models had been established for virus replication. Human stool samples obtained from outbreaks and from human volunteer studies were the only source of the virus, yet the concentration of the virus in stool is so low that virus detection with routine electron microscopy was not possible. However, the recently successful expression of NOR capsid proteins by baculoviruses (double stranded DNA viruses which infect mainly insects) in insect cells has provided a valuable alternative for studying the immunology, epidemiology and pathogenesis of NORs. The viral capsid protein monomers produced self-assemble into virus-like particles (VLPs). These VLPs are morphologically and antigenically indistinguishable from authentic viruses found in human feces, providing a useful tool for the development of immunological assays and the study of receptor-virus interaction.
The atomic structure of the recombinant NOR capsid protein indicates that it contains 180 capsid protein monomers organized into 90 dimeric capsomers that form a T=3 icosahedron. Data from cryoelectron microscopy and X-ray crystallography showed that the viral capsid protein folds into two major domains, the N-terminal Shell (S) domain and the C-terminal Protrusion (P) domain. The S-domain forms the interior shell, while the P-domain builds up arch-like structures that extend from or protrude from the shell. Morphogenesis studies showed that the S-domain contains elements required for assembly of the interior shell of the capsid, whereas intermolecular contacts between dimeric subunits of the P-domain increase the stability of the capsid. These two domains are linked through a 8-10-residue (amino acid) hinge. The P-domain is further divided into P1 and P2 domains, with the latter located at the most exterior surface of the capsid. In contrast to the S and P1 domains, the P2-domain has a high sequence variation. Since the P-domain is located at the most exterior surface of the viral particle and contains the most variable sequence, it is believed that the P-domain is responsible for host interactions, immune recognition, receptor binding and immune responses. It has been shown that isolated P-domains having a hinge (but lacking the S-domain) form dimers in vitro that maintain binding to human histo-blood group antigen (HBGA) receptors.
HBGAs are a heterogeneous group of complex glycans and related carbohydrates. NORs recognize human HBGAs as receptors in a diverse, strain-specific manner. Among the HBGAs, the most commonly encountered blood groups are ABO (ABH) and Lewis. The biosynthetic pathways used in forming antigens in the ABH and Lewis blood group systems are interrelated.
Human HBGAs are present on many cell types including red blood cells and vascular endothelial cells, as well as on the mucosal epithelia of the gastrointestinal, uro-genital and respiratory tracts. They can also be present in a soluble form in biologic fluids such as blood, saliva, gastrointestinal contents and milk. HBGAs are synthesized from a series of precursor structures by stepwise addition of monosaccharide units via a set of glycosyltransferases that are genetically controlled and known as the ABO, Lewis, and secretor gene families.
The human HBGA system is highly polymorphic and is controlled by multiple gene families with silent alleles. The presence of such diversified molecules as HBGAs on the cell surfaces indicates a possible host defense mechanism against the changing external environment. Nevertheless, HBGAs have been linked to infection by several bacterial and viral pathogens, and may provide a “docking station” for noroviruses. That is, HBGAs can be a recognition target for pathogens and may facilitate entry into a cell that expresses or forms a receptor-ligand bond with the antigens. While the exact nature of such an interaction is not currently known, close association of a pathogen that would occur with antigen binding may play a role in anchoring the pathogen to the cell as an initial step in the infection process.
The recognition of human HBGAs by NORs is a typical protein-carbohydrate interaction, in which the protruding domain of the viral capsid protein forms an interface with the oligosaccharide side-chains of the HBGA antigens, with a wide diversity among different strains. As pathogens that replicate possibly only in the intestinal tract, NORs have developed unique strategies to overcome the host defense system. This has been shown by their genetic and structural variations, which explains why NOR-associated diseases are so common and widespread in every population worldwide.
PCT Patent Publication US2003/101176, published Dec. 2, 2003, which is incorporated herein by reference in its entirety, relates to the binding of NOR strains to ABO and Lewis HBGAs in one of several distinct histo-blood group patterns. The recognition of HBGAs by NORs is strain specific, and a number of distinct HBGA binding patterns have been identified. More binding patterns may be found, based on the diversity of NORs and the polymorphism of carbohydrates on host cell surfaces.
PCT Patent Publication US2006/138,514, published Dec. 28, 2006, which is incorporated herein by reference in its entirety, relates to a small particle, known as the P-particle, which displays enhanced binding affinity to HBGAs. The P-particle is a T=1 icosahedron built by 24 P-domain monomers that organize into 12 identical P-domain dimers. Both 12 (P-domain dimers) and 24 (P-domain monomers) are perfect unit numbers for an icosahedral symmetry that occurs frequently for plant and animal viruses. The isolated P-domain, without the S-domain or the hinge of the monomer capsid protein, can spontaneously form a T=1 icosahedral P-particle, a complex consisting of 24 P-domain monomers arranged into 12 dimers. The P-particle can bind to the corresponding HBGAs and reveals strong blocking of NOR VLP binding to the HBGAs. The spontaneous formation of P-particles has been observed with various strains of NOR, including strains VA387, MOH, and Norwalk Virus (NV). The NOR P-particle is useful in the therapeutic treatment of the NOR infection, and in creating a vaccine against NOR infection.
Both Rotaviruses (RVs) and NORs are common pathogens worldwide that incur a large burden of disease. On a worldwide basis, up to 1 billion episodes of gastroenteritis of all causes occur each year in children <5 years of age, of which 13 to 25% (˜130 million episodes) are caused by RVs. RVs are the leading cause of severe diarrhea and dehydration among children and each year severe RV gastroenteritis causes 350,000-600,000 deaths in children <5 years of age. It also accounts for 2 million childhood hospital admissions with an estimated cost of over 1 billion dollars per year. On the other hand, NORs are the most important cause of non-bacterial epidemics of acute gastroenteritis, affecting individuals of all ages. NORs are highly contagious and can be spread quickly leading to large outbreaks in a variety of settings. A recent report estimated that NORs cause 1,091,000 inpatient hospitalizations and 218,000 deaths in children <5 years of age in the developing countries each year. In the USA foodborne pathogens infect an estimated 76 million people each year and are the cause of 325,000 hospitalizations. NORs alone cause $350 to $750 million in losses each year due to clinical care and lost revenue from recalled foods.
Although two new RV vaccines (Rotarix™, GlaxoSmithKline and RotaTeq®, Merck) have recently been introduced there are several issues related to the vaccine that are not yet fully resolved: a) its efficacy when vaccinated and non-vaccinated children are exposed to a wider range of RV serotypes than those found in the vaccines; b) vaccine cost and distribution costs leading to questions of how widely these vaccines will be distributed into poor countries where they are most needed; and c) the level of protection in developing countries, where mortality is highest, is still being determined. These live attenuated vaccines could possible revert or reassort to produce virulent strains. Thus, there is a need for a new generation of subunit vaccines containing highly effective neutralizing epitopes of RV. Currently, there is no treatment for NOR-associated diseases. Therefore, the development of an effective vaccine against NOR and RV, especially a single vaccine that could protect against both, would fulfill a major clinical need, further emphasizing the significant commercial potential of the P-particle vaccine platform disclosed herein.
Notwithstanding the advancements in the therapeutic treatment of and a vaccine development against NOR infections, there remains a need for improving the identification of infections caused by other virus types, the therapeutic treatment of other virus types, vaccine development against other virus types, and the development of an improved drug delivery system to target a specific tissue or organ.